Detection of Minute virus of mice strains in different cell lines: Implications for adventitious agent testing.

Minute virus of mice (MMV) has contaminated biotechnological processes in the past and specific MMV testing is therefore recommended, if the production cell line is known to be permissive for this virus. Testing is widely done using cell-culture-based adventitious virus assays, yet MMV strains may differ in their in vitro cell tropism. Here, we investigated the growth characteristics of different MMV strains on A9 and 324K cells and identified significant differences in susceptibility of these widely used indicator cell lines to infection by different strains of MMV, which has implications for MMV detectability during routine testing of biotechnology process harvests.

Optimization of an Antibody Light Chain Framework Enhances Expression, Biophysical Properties and Pharmacokinetics.

Efficacy, safety, and manufacturability of therapeutic antibodies are influenced by their biopharmaceutical and biophysical properties. These properties can be optimized by library approaches or rationale protein design. Here, we employed a protein engineering approach to modify the variable domain of the light chain (VL) framework of an oxidized macrophage migration inhibitory factor (oxMIF)-specific antibody.

A nonenveloped virus with a lipid envelope: hepatitis A virus as used in virus-reduction studies.

Background: Recently, a quasi-lipid-enveloped (LE) form of the traditionally nonlipid-enveloped (NLE) hepatitis A virus (HAV) was described in human serum and cell culture-derived HAV stocks. This discovery challenges the understanding of HAV reduction in virus clearance studies of plasma products, which were performed under the premise of an NLE nature of this virus. Here, the presence of LE particles in HAV stocks used for reduction studies was verified, and the hypothesis that LE and NLE particles might contribute to the differential heat sensitivity of HAV variants during heat treatment of human serum albumin was evaluated.

Hepatitis E virus and the safety of plasma products: investigations into the reduction capacity of manufacturing processes.

Background: Hepatitis E virus (HEV) has been transmitted by transfusion of labile blood products and the occasional detection of HEV RNA in plasma pools indicates that HEV viremic donations might enter the manufacturing process of plasma products. To verify the safety margins of plasma products with respect to HEV, virus reduction steps commonly used in their manufacturing processes were investigated for their effectiveness to reduce HEV.

Study Design And Methods: Detection methods for HEV removal (by reverse transcription quantitative polymerase chain reaction) and inactivation (using an infectivity assay) were established.

Development of a transgenic mouse model with immune tolerance for human coagulation factor VIIa.

Purpose: Human factor VIIa (FVIIa) is commonly used as bypassing therapy to treat bleeding episodes in hemophilia patients with neutralizing antibodies to factors VIII (FVIII) or IX (FIX). There is a need for a suitable animal model to assess the immunogenicity of new FVIIa products during preclinical development. The aim of this study was the design of a novel transgenic mouse model with immune tolerance to human FVIIa.

Human anti-macrophage migration inhibitory factor antibodies inhibit growth of human prostate cancer cells in vitro and in vivo.

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine, originally discovered for its eponymous effect and now known for pleiotropic biologic properties in immunology and oncology. Circulating MIF levels are elevated in several types of human cancer including prostate cancer. MIF is released presumably by both stromal and tumor cells and enhances malignant growth and metastasis by diverse mechanisms, such as stimulating tumor cell proliferation, suppressing apoptotic death, facilitating invasion of the extracellular matrix, and promoting angiogenesis.

Neutralization of macrophage migration inhibitory factor (MIF) by fully human antibodies correlates with their specificity for the β-sheet structure of MIF.

The macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine that recently emerged as an attractive therapeutic target for a variety of diseases. A diverse panel of fully human anti-MIF antibodies was generated by selection from a phage display library and extensively analyzed in vitro. Epitope mapping studies identified antibodies specific for linear as well as structural epitopes.

Maintenance and break of immune tolerance against human factor VIII in a new transgenic hemophilic mouse model.

Replacement of the missing factor VIII (FVIII) is the current standard of care for patients with hemophilia A. However, the short half-life of FVIII makes frequent treatment necessary. Current efforts focus on the development of longer-acting FVIII concentrates by introducing chemical and genetic modifications to the protein.

Modulation of ADAMTS13 secretion and specific activity by a combination of common amino acid polymorphisms and a missense mutation.

Sequence analysis of the ADAMTS13 locus of 2 patients with hereditary thrombotic thrombocytopenic purpura (TTP) revealed the homozygous presence of 4 single nucleotide polymorphisms (SNPs) (R7W, Q448E, P618A, A732V) and a rare missense mutation (R1336W). Analysis of the individual effect of any amino acid exchanges showed that several sequence variations can interact with each other, thereby altering the phenotype of ADAMTS13 deficiency. Introduction of polymorphisms R7W, Q448E, and A732V had no or only minor effects on ADAMTS13 secretion.

Fatal congenital thrombotic thrombocytopenic purpura with apparent ADAMTS13 inhibitor: in vitro inhibition of ADAMTS13 activity by hemoglobin.

Severe ADAMTS13 deficiency in thrombotic thrombocytopenic purpura (TTP) is either constitutional and caused by ADAMTS13 mutations, or acquired and most often due to ADAMTS13 inhibitory autoantibodies. In strongly hemolytic serum of a pediatric patient, diagnosed with TTP postmortem, ADAMTS13 activity was less than 3%. Both parents had an ADAMTS13 activity of approximately 50%.

ADAMTS13 gene defects in two brothers with constitutional thrombotic thrombocytopenic purpura and normalization of von Willebrand factor-cleaving protease activity by recombinant human ADAMTS13.

Genetic analysis of the ADAMTS13 locus identified six mutations in the ADAMTS13 genes of two brothers suffering from constitutional thrombotic thrombocytopenic purpura (TTP): a stop codon leading to a truncated protein on the paternal ADAMTS13 allele and five amino acid exchanges on the maternal allele, three of which were single nucleotide polymorphisms. The other two mutations, not detected in 230 sequenced alleles of healthy control subjects, are, therefore, probably responsible, alone or as part of a combination, for the severe ADAMTS13 deficiency. We also investigated the feasibility of using recombinant ADAMTS13 (rADAMTS13) for normalization of von Willebrand factor-cleaving protease (VWF-cp) activity in plasma of the two congenitally deficient patients.

Cloning, expression, and functional characterization of the von Willebrand factor-cleaving protease (ADAMTS13).

Deficient von Willebrand factor (VWF) degradation has been associated with thrombotic thrombocytopenic purpura (TTP). In hereditary TTP, the specific VWF-cleaving protease (VWF-cp) is absent or functionally defective, whereas in the nonfamilial, acquired form of TTP, an autoantibody inhibiting VWF-cp activity is found transiently in most patients. The gene encoding for VWF-cp has recently been identified as a member of the metalloprotease family and designated ADAMTS13, but the functional activity of the ADAMTS13 gene product has not been verified.